Patient-derived monoclonal antibody neutralizes SARS-CoV-2 Omicron variants and confers full protection in monkeys.

The SARS-CoV-2 Omicron variant has very high levels of transmission, is resistant to neutralization by authorized therapeutic human monoclonal antibodies (mAb) and is less sensitive to vaccine-mediated immunity. To provide additional therapies against Omicron, we isolated a mAb named P2G3 from a previously infected vaccinated donor and showed that it has picomolar-range neutralizing activity against Omicron BA.1, BA.1.1, BA.2 and all other variants tested. We solved the structure of P2G3 Fab in complex with the Omicron spike using cryo-electron microscopy at 3.04 Å resolution to identify the P2G3 epitope as a Class 3 mAb that is different from mAb-binding spike epitopes reported previously. Using a SARS-CoV-2 Omicron monkey challenge model, we show that P2G3 alone, or in combination with P5C3 (a broadly active Class 1 mAb previously identified), confers complete prophylactic or therapeutic protection. Although we could select for SARS-CoV-2 mutants escaping neutralization by P2G3 or by P5C3 in vitro, they had low infectivity and 'escape' mutations are extremely rare in public sequence databases. We conclude that this combination of mAbs has potential as an anti-Omicron drug.

ACE2-binding exposes the SARS-CoV-2 fusion peptide to broadly neutralizing coronavirus antibodies.

The coronavirus spike glycoprotein attaches to host receptors and mediates viral fusion. Using a broad screening approach, we isolated seven monoclonal antibodies (mAbs) that bind to all human-infecting coronavirus spike proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune donors. These mAbs recognize the fusion peptide and acquire affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha- and betacoronaviruses, including animal coronaviruses WIV-1 and PDF-2180. Two selected mAbs also neutralize Omicron BA.1 and BA.2 authentic viruses and reduce viral burden and pathology in vivo. Structural and functional analyses showed that the fusion peptide-specific mAbs bound with different modalities to a cryptic epitope hidden in prefusion stabilized spike, which became exposed upon binding of angiotensin-converting enzyme 2 (ACE2) or ACE2-mimicking mAbs.

ALG-097111, a potent and selective SARS-CoV-2 3-chymotrypsin-like cysteine protease inhibitor exhibits in vivo efficacy in a Syrian Hamster model.

There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 µM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.

Pan-viral protection against arboviruses by activating skin macrophages at the inoculation site.

Arthropod-borne viruses (arboviruses) are important human pathogens for which there are no specific antiviral medicines. The abundance of genetically distinct arbovirus species, coupled with the unpredictable nature of their outbreaks, has made the development of virus-specific treatments challenging. Instead, we have defined and targeted a key aspect of the host innate immune response to virus at the arthropod bite that is common to all arbovirus infections, potentially circumventing the need for virus-specific therapies. Using mouse models and human skin explants, we identify innate immune responses by dermal macrophages in the skin as a key determinant of disease severity. Post-exposure treatment of the inoculation site by a topical TLR7 agonist suppressed both the local and subsequent systemic course of infection with a variety of arboviruses from the Alphavirus, Flavivirus, and Orthobunyavirus genera. Clinical outcome was improved in mice after infection with a model alphavirus. In the absence of treatment, antiviral interferon expression to virus in the skin was restricted to dermal dendritic cells. In contrast, stimulating the more populous skin-resident macrophages with a TLR7 agonist elicited protective responses in key cellular targets of virus that otherwise proficiently replicated virus. By defining and targeting a key aspect of the innate immune response to virus at the mosquito bite site, we have identified a putative new strategy for limiting disease after infection with a variety of genetically distinct arboviruses.

Glutathione is a highly efficient thermostabilizer of poliovirus Sabin strains.

Glutathione (GSH) is the most abundant thiol peptide in animal cells and has a critical role in antioxidation. GSH was reported to be essential for stabilization of some enteroviruses, including poliovirus (PV), during viral morphogenesis. Here, we explored the potential use of GSH as a thermostabilizer of oral poliomyelitis vaccine (OPV) formulations. GSH significantly protected the three types of PV from heat-inactivation in a concentration-dependent manner. At a GSH concentration of 20mM, nearly complete protection was observed against heating temperatures up to 53°C for 2min.GSH also markedly protected PV1 from heat-inactivation and this up to 6 h at temperatures of 44°C and 46°C and 3 h at 48°C. The fact that GSH is naturally present at high concentration in the human body makes it an efficient candidate stabilizer for OPV formulations.

Protein kinases C as potential host targets for the inhibition of chikungunya virus replication.

We have shown previously that prostratin, a non-tumor promoting phorbol ester, inhibits chikungunya virus (CHIKV)-induced cytopathic effects in vitro. Prostratin is a potent activator of protein kinases C (PKC), a family of related serine/threonine kinases that regulate many cellular processes such as proliferation and apoptosis. The objective of this study was to explore the mechanism of the anti-CHIKV activity of prostratin. Prostratin reduced the production of infectious virus particles and viral protein accumulation in a dose-dependent manner at a post-entry step during virus replication. The antiviral effect of the compound was cell-dependent, with potent antiviral activity observed in human skin fibroblasts cells, the primary target cells of CHIKV infection. The antiviral activity of prostratin was markedly reduced in the presence of PKC inhibitors, therefore confirming that the antiviral effect results from an activation of PKCs. Together these results showed that PKCs are potential host targets for the inhibition of CHIKV replication.

Discovery of novel multi-target indole-based derivatives as potent and selective inhibitors of chikungunya virus replication.

We recently identified indole derivatives (IIIe and IIIf) with anti-chikungunya virus (CHIKV) activities at lower micro molar concentrations and a selective index of inhibition higher than the lead compound Arbidol. Here we highlight new structural information for the optimization of the previously identified lead compounds that contain the indole chemical core. Based on the structural data, a series of indole derivatives was synthesized and tested for their antiviral activity against chikungunya virus in Vero cell culture by a CPE reduction assay. Systematic optimization of the lead compounds resulted in tert-butyl-5-hydroxy-1-methyl-2-(2-trifluoromethysulfynyl)methyl)-indole-3-carboxylate derivative IIc with a 10-fold improved anti-CHIKV inhibitory activity (EC50=6.5±1µM) as compared to Arbidol demonstrating a potent, selective and specific inhibition of CHIKV replication with only a moderate cell protective effect against other related alphaviruses. The reported computational insights, together with the accessible synthetic procedure, pave the road towards the design of novel synthetic derivatives with enhanced anti-viral activities.

Simplified Bryostatin Analogues Protect Cells from Chikungunya Virus-Induced Cell Death.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus showing a recent resurgence and rapid spread worldwide. While vaccines are under development, there are currently no therapies to treat this disease, except for over-the-counter (OTC) analgesics, which alleviate the devastating arthritic and arthralgic symptoms. To identify novel inhibitors of the virus, analogues of the natural product bryostatin 1, a clinical lead for the treatment of cancer, Alzheimer's disease, and HIV eradication, were investigated for in vitro antiviral activity and were found to be among the most potent inhibitors of CHIKV replication reported to date. Bryostatin-based therapeutic efforts and even recent anti-CHIKV strategies have centered on modulation of protein kinase C (PKC). Intriguingly, while the C ring of bryostatin primarily drives interactions with PKC, A- and B-ring functionality in these analogues has a significant effect on the observed cell-protective activity. Significantly, bryostatin 1 itself, a potent pan-PKC modulator, is inactive in these assays. These new findings indicate that the observed anti-CHIKV activity is not solely mediated by PKC modulation, suggesting possible as yet unidentified targets for CHIKV therapeutic intervention. The high potency and low toxicity of these bryologs make them promising new leads for the development of a CHIKV treatment.

Inhibition of Chikungunya Virus-Induced Cell Death by Salicylate-Derived Bryostatin Analogues Provides Additional Evidence for a PKC-Independent Pathway.

Chikungunya virus (CHIKV) has been spreading rapidly, with over one million confirmed or suspected cases in the Americas since late 2013. Infection with CHIKV causes devastating arthritic and arthralgic symptoms. Currently, there is no therapy to treat this disease, and the only medications focus on relief of symptoms. Recently, protein kinase C (PKC) modulators have been reported to inhibit CHIKV-induced cell death in cell assays. The salicylate-derived bryostatin analogues described here are structurally simplified PKC modulators that are more synthetically accessible than the natural product bryostatin 1, a PKC modulator and clinical lead for the treatment of cancer, Alzheimer's disease, and HIV eradication. Evaluation of the anti-CHIKV activity of these salicylate-derived bryostatin analogues in cell culture indicates that they are among the most potent cell-protective agents reported to date. Given that they are more accessible and significantly more active than the parent natural product, they represent new therapeutic leads for controlling CHIKV infection. Significantly, these analogues also provide evidence for the involvement of a PKC-independent pathway. This adds a fundamentally distinct aspect to the importance or involvement of PKC modulation in inhibition of chikungunya virus replication, a topic of recent and growing interest.